Oestrogen-dependent hypothalamic oxytocin expression with changes in feeding and body weight in female rats

Oxytocin (OXT) is produced in the hypothalamic nuclei and secreted into systemic circulation from the posterior pituitary gland. In the central nervous system, OXT regulates behaviours including maternal and feeding behaviours. Our aim is to evaluate whether oestrogen regulates hypothalamic OXT dynamics. Herein, we provide the first evidence that OXT dynamics in the hypothalamus vary with sex and that oestrogen may modulate dynamic changes in OXT levels, using OXT-mRFP1 transgenic rats. The fluorescence intensity of OXT-mRFP1 and expression of the OXT and mRFP1 genes in the hypothalamic nuclei is highest during the oestrus stage in female rats and decreased significantly in ovariectomised rats. Oestrogen replacement caused significant increases in fluorescence intensity and gene expression in a dose-related manner. This is also demonstrated in the rats’ feeding behaviour and hypothalamic Fos neurons using cholecystokinin-8 and immunohistochemistry. Hypothalamic OXT expression is oestrogen-dependent and can be enhanced centrally by the administration of oestrogen.

1. Several recent studies have also examined the interaction between estrogen and oxytocin in the control of food intake. I think a discussion of previous other literature ( PMID: 31738883, 25647756, 30118729) and how your findings fit in with these studies would be beneficial.
2. The authors found that estrogen-induced oxytocin neurons in the PVH, SON and PPG. Within these areas are both magnocellular neurons and parvocellular neurons that have differing projections. Do the authors think estrogen-induced oxytocin activity in periphery and brain are same neurons, different, or interacting? 3. While the authors showed a clear estrogen-induced increase in oxytocin neuron activity (via fos), have the authors examined binding affinity or oxytocin receptor expression? 4. I think a direct measure of an interaction between estrogen and oxytocin is necessary to conclude that oxytocin is dependent on estrogen for food intake control. The authors could try subthreshold doses of both oxytocin and estrogen. If there is a combined inhibitory effect this would demonstrate an interaction.
Reviewer #3 (Remarks to the Author): Using OXT-mRFP1 transgenic rats, the first half of this manuscript describes changes in the fluorescence intensity of oxytocin reporter fluorescence protein (OXT-mRFP1) in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and posterior pituitary during estrous cycle in intact females, as well as in ovariectomized (OVX) females and OVX females received estradiol (E2) treatment.
The second half of the manuscript describes the effects of OVX and E2 treatment on body weight and food intake in longer term, as well as the short-term effect of ip injection of CCK-8 and icv infusion of oxytocin antagonist on food intake in OVX with/without E2 treated females.
From these results, authors concluded that: 1) oxytocin expression in the hypothalamus is estrogen dependent; and 2) suppression of food intake by estrogen may be mediated by anorexigenic activity of oxytocin.
There is a number of major deficiencies and flaws in experimental design and interpretation of results.

Major concerns:
The title does not represent the content of the manuscript.
Assessment of oxytocin production in the hypothalamus and pituitary is solely by the fluorescence intensity of reporter protein. Was the fluorescence intensity correlated with the amount of oxytocin? Additional methods to validate, such as qPCR of mRNA extracted from punched tissue and/or quantitative in situ hybridization, are required.
Material and Methods does not adequately address how the fluorescence intensity was measured. It is hard for a reviewer to assess the competence of the experimental design and methods.
1. The SON in rats extends anterior to posterior direction in about 1 mm. If the brain sections were cut at 30 µm thickness, there are ~33 sections that containing the SON. Were the measurements obtained from every section or just a few sections? The size and shape of the SON changes from anterior to posterior. More importantly for the same context, the population (density) of oxytocin neurons within the of the SON varies considerably section to section. This fact is especially important when the fluorescence intensity of brain areas rather than oxytocin neurons themselves was obtained.
2. What is the reason for measuring the fluorescence intensity of brain areas rather than oxytocin neurons? What are the limitations and reasonable interpretation of the results? 3. How were the "% of control" obtained? If a normalization method was used, it must be addressed adequately. Were the exposure time and intensity of light equal to all images obtained? Questionable interpretation of the results.
1. Oxytocin expression in the hypothalamus is NOT estrogen dependent. In fact, OVX females still express oxytocin in the hypothalamus.
2. The notion addressed by the authors "suppression of food intake by estrogen may be mediated by anorexigenic activity of oxytocin" is probable, but extremely far-fetched considering the wide distribution of oxytocin receptor (OXTR) and estrogen receptors in the nervous system (both peripheral and central) and peripheral organs. a) OXTR are widely distributed in the brain, including the hypothalamic area (not SON or PVN) that regulate food intake. Therefore, the icv infusion of OTA affects numbers of OXTR in the brain. Moreover, the expression of OXTR in some areas of the brain are totally estrogen dependent. b) Estrogen receptors are widely distributed in the hypothalamus especially in the area regulating food intake. Estrogen receptors are also found in the peripheral tissues including the adipose tissue.
c) CCK receptors are widely located in the brain and peripheral organ such as the intestine. Thus, ip injection of CCK-8 would affect any of the brain areas and peripheral organs, not just oxytocin neurons.
Due to the major concerns addressed above, additional experiments and an extensive revision of the manuscript are required. For these reasons, I do not recommend the manuscript as in the current form for publication in Nature Communication.

Responses to the reviewers' comments Manuscript No.: COMMSBIO-21-0650
Title: Oestrogen-dependent hypothalamic oxytocin expression with changes in feeding and body weight in female rats Journal Title: Communications biology, Submission Date: 13th March 2021

Reviewer #1 (Remarks to the Author):
We would like to thank the reviewer for their robust and detailed assessment of our manuscript. The manuscript has been carefully revised according to the suggestions and recommendations.
The title could be more "pro-active" and describe the new discoveries of the studies. Response: We thank the reviewer for their valuable suggestion. We have revised the title to "Oestrogen-dependent hypothalamic oxytocin expression with changes in feeding and body weight in female rats" accordingly.
The abstract should include the CCK results. Response: We thank the reviewer for their comment. We have revised the abstract accordingly (line 33).
Is it correct that the authors used a separate cohort of females for every experiment-i.e., they never correlated the OXT-mRFP1 fluorescence with feeding? This seems like a missed opportunity. Response: We thank the reviewer for their pertinent comment. We used different rats in each of these experiments. The body weight experiment required the use of anesthesia for micro-CT and did not use OXT-mRFP1 rats with varying fluorescence.
A one-way ANOVA for statistical analysis for Experiments 5-7 seems inappropriate since the authors are looking at group differences over time and comparing drug (CCK and OTX antagonist) effects. A two-way ANOVA would seem to be more appropriate. Response: We thank the reviewer for their comment and apologise for this error. We have included details of a two-way ANOVA and repeated-measures ANOVA in the statistics section of the revised manuscript (Lines 399, 410, and 597).
It would help to focus the Discussion on the role of the anorexigenic hormones E2 and CCK in inhibiting food intake via the OXT signaling network. A simple circuit model would help drive home the point.

Response:
We thank the reviewer for their constructive suggestion.
Minor points: 1. Line 237, should be "OXT neurons." Response: We thank the reviewer for their suggestion. We have revised the sentence accordingly (Line 267) .
2. Line 267, should be "females compared with male rats;" and "females at prooestrous, metoestrous, dioestrous stages compared with oestrous female rats." Response: We thank the reviewer for their constructive suggestion. We have revised the sentence accordingly (Line 333, 334) 3. Line 463, should be "goat anti-cFos." Response: We thank the reviewer for their pertinent suggestions and apologise for the confusion. We have revised the manuscript accordingly (Line 585).

Reviewer #2 (Remarks to the Author):
We would like to thank the reviewer for their robust and detailed assessment of our manuscript. The manuscript has been carefully revised according to the suggestions and recommendations.

Several recent studies have also examined the interaction between estrogen and oxytocin in
the control of food intake. I think a discussion of previous other literature (PMID: 31738883, 25647756, 30118729) and how your findings fit in with these studies would be beneficial. Response: We thank the reviewer for their constructive suggestion. We have added one reference and revised the manuscript accordingly (Lines 280-284).

The authors found that estrogen-induced oxytocin neurons in the PVH, SON and PPG.
Within these areas are both magnocellular neurons and parvocellular neurons that have differing projections. Do the authors think estrogen-induced oxytocin activity in periphery and brain are same neurons, different, or interacting? Response: We thank the reviewer for their constructive comment. In this experiment, as shown in Fig. 2-4, the oestrus cycle and oestrogen affected both magnocellular neurons and parvocellular oxytocin neurons of the PVN and SON, leading to a similar result in both sets of neurons. However, it is unclear whether they are the same neurons or interact with each other.

While the authors showed a clear estrogen-induced increase in oxytocin neuron activity (via fos), have the authors examined binding affinity or oxytocin receptor expression? Response:
We thank the reviewer for their important comment. This study was unable to determine the binding affinity or expression of the oxytocin receptor. We believe that this is a future research topic. We have revised the discussion section to reflect this (Lines 289-290).

I think a direct measure of an interaction between estrogen and oxytocin is necessary to
conclude that oxytocin is dependent on estrogen for food intake control. The authors could try subthreshold doses of both oxytocin and estrogen. If there is a combined inhibitory effect this would demonstrate an interaction. Response: We thank the reviewer for their constructive suggestion. We measured the subthreshold doses of both CCK-8 (0.5 μg/ml.) and oestrogen (8 μg β-oestradiol/ml sesame oil). However, examination of the interaction at subthreshold doses did not reveal any inhibitory effect. We also performed additional experiments which also revealed no inhibitory effect.

Reviewer #3 (Remarks to the Author):
We would like to thank you for your robust and detailed assessment of our manuscript. The manuscript has been carefully revised in line with your suggestions and recommendations.
The title does not represent the content of the manuscript. Response: We thank the reviewer for their pertinent comment. We have revised the title accordingly ("Oestrogen-dependent hypothalamic oxytocin expression with changes in feeding and body weight in female rats".)

Response:
We thank the reviewer for their constructive comment. We have added details regarding in situ hybridization histochemical examination with OXT-mRFP1 mRNA in the revised manuscript.
We have accordingly revised the title, introduction section, and results section.
Material and Methods does not adequately address how the fluorescence intensity was measured. It is hard for a reviewer to assess the competence of the experimental design and methods. Response: We thank the reviewer for their constructive comment and apologise for the misunderstanding. We have deleted this sentence and revised the manuscript accordingly (Lines 518-531). section. This fact is especially important when the fluorescence intensity of brain areas rather than oxytocin neurons themselves was obtained. Response: We thank the reviewer for their pertinent comments and apologise for the confusion. The tissue was cut into 30-μm-thick sections with a microtome for observation. The sections were divided into three groups, so that approximately the same brain region was included. Using the first group of sections, mRFP1 fluorescence in the hypothalamus was observed and photographed using a fluorescence microscope. We have revised the manuscript accordingly (Lines 548-550).

The SON in rats extends
2. What is the reason for measuring the fluorescence intensity of brain areas rather than oxytocin neurons? What are the limitations and reasonable interpretation of the results? Response: We thank the reviewer for their pertinent questions. In this study, we quantitatively evaluated OXT expression in OXT-mRFP1 transgenic rats by measuring fluorescence intensity as reported previously (Arase et al., 2018;Katoh et al., 2011). Therefore, the fluorescence intensity was measured and quantitatively evaluated.
3. How were the "% of control" obtained? If a normalization method was used, it must be addressed adequately. Were the exposure time and intensity of light equal to all images obtained? Response: We thank the reviewer for their important comment. Using a light source of the same intensity, we averaged the mRFP1 fluorescence intensities for each region. We have clarified this in lines (Lines 559-560).
Questionable interpretation of the results.

Oxytocin expression in the hypothalamus is NOT estrogen dependent. In fact, OVX females
still express oxytocin in the hypothalamus. Response: We thank the reviewer for their comment. Oestrogen regulates changes in OXT expression in hypothalamic OXT neurons in a dose-dependent manner. To avoid confusion, we have changed the wording to reflect this in line (Line 32 and 132).
2. The notion addressed by the authors "suppression of food intake by estrogen may be mediated by anorexigenic activity of oxytocin" is probable, but extremely far-fetched considering the wide distribution of oxytocin receptor (OXTR) and estrogen receptors in the nervous system (both peripheral and central) and peripheral organs. a) OXTR are widely distributed in the brain, including the hypothalamic area (not SON or PVN) that regulate food intake. Therefore, the icv infusion of OTA affects numbers of OXTR in the brain. Moreover, the expression of OXTR in some areas of the brain are totally estrogen dependent. Response: We thank the reviewer for their important comments. This study did not examine oxytocin or oestrogen receptors. It is unknown whether OXTR expression is oestrogen-dependent.
We believe that this is worthy of investigation in future studies. b) Estrogen receptors are widely distributed in the hypothalamus especially in the area regulating food intake. Estrogen receptors are also found in the peripheral tissues including the adipose tissue. Response: We thank the reviewer for their helpful comments. Oestrogen receptors are also found in peripheral tissues, including adipose tissue. Therefore, we believe that there is a change in body weight. However, this study did not examine the oestrogen receptor. It is stated in the limitation (Lines 289-290). c) CCK receptors are widely located in the brain and peripheral organ such as the intestine.
Thus, ip injection of CCK-8 would affect any of the brain areas and peripheral organs, not just oxytocin neurons. Response: We thank the reviewer for their constructive suggestion. As the reviewer has correctly mentioned, CCK receptors are widely present in peripheral organs such as the brain and intestine.
Therefore, various effects are expected. However, in this experiment, the relevant examinations were performed under the same conditions whereby CCK-8 was administered.